1. Volume 12, Issue 2, Pages 187-199 (August 2012)
Translocation of Sickle Cell Erythrocyte MicroRNAs into Plasmodium falciparum Inhibits Parasite Translation and Contributes to Malaria Resistance 
Gregory LaMonte, Nisha Philip, Joseph Reardon, Joshua R. Lacsina, William Majoros, Lesley Chapman, Courtney D. Thornburg, Marilyn J. Telen, Uwe Ohler, Christopher V. Nicchitta, Timothy Haystead, Jen-Tsan Chi 
Cell Host & Microbe 
Volume 12, Issue 2, Pages (August 2012)
DOI: /j.chom
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2. Figure 1 Erythrocyte miRNAs Are Translocated into P. falciparum
(A) The 11 most abundant miRNAs found in the IDC of P. falciparum and their percentage representation in either uninfected erythrocytes or P. falciparum at 8 or 32 hr after infection.
(B) Levels of the three indicated miRNAs shown in parasites at 8, 16, 32, and 40 hr after infection.
(C) Relative levels of erythrocytic miR-451 in untreated HbAA, HbAS, and HbSS erythrocytes (normalized by cell number, n = 6; mean ± SEM).
(D) Relative levels of erythrocytic miR-451 in untreated HbAA, HbAS, and HbSS erythrocytes and with indicated treatments (normalized by cell number, n = 6; mean ± SEM).
(E) Detection of miRNA localization in uninfected and infected (saponin-lysed) erythrocytes indicating transfected miRNA (green), the PVM (EXP1, red), and parasite nuclei (DAPI, blue).
See also Figure S1 and Table S1.
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3. Figure 2
Elevated miRNA Levels Confer Resistance to P. falciparum in HbS Erythrocytes
(A) Normalized parasitemia at different time points after infection in HbAA erythrocytes transfected with control DNA or the indicated miRNAs (n = 24; mean ± SEM, except miR-16 and let-7i, which are n = 8).
(B) Parasite proliferation measured by [3H]-hypoxanthine incorporation in HbAA erythrocytes transfected with the indicated miRNAs (n = 4; mean ± SEM).
(C) Intraparasitic miR-451 levels in parasites grown in untreated HbAA, HbAS, or HbSS erythrocytes, HbAA erythrocytes transfected with the indicated miRNA, and HbSS erythrocytes transfected with antisense 2′O-methyl oligonucleotides, normalized against the total cell number and HbSS (n = 6; mean ± SEM).
(D) Normalized growth rates of parasites propagated in HbAA, HbAS, or HbSS erythrocytes transfected with the indicated antisense 2′O-methyl oligonucleotides to inhibit specific miRNAs (n = 18 except for HbSS miR-451+miR-223, where n = 5 and in all HbAS samples where n = 6; mean ± SEM).
See also Figure S2.
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4. Figure 3
Human miR-451 Forms Chimeric Fusion RNAs with P. falciparum mRNAs
(A) Northern blot of total RNA from human mammary epithelial cells (HMEC), blood, synthetic miR-451, and purified parasite (3D7) probed for miR-451 or miR-181 via LNA probe. Amounts of RNA were visualized by ethidium bromide staining.
(B) Northern blot of polyA+ (upper-middle panel) RNA from HMEC, blood, synthetic miR-451, and purified parasite (3D7) were probed for miR-451 (upper panel) and PKA-R (lower panel). The postulated size of the miR-451-PKA-R is indicated with a star, while the expected positions of the Plasmodium rRNA are indicated with arrows.
(C) Northern blot of total RNA from whole blood and purified parasite (3D7) derived from HbAA, HbAS, and HbSS cells probed for miR-451. RNA amounts were visualized by ethidium bromide staining. The two bands indicated with “a” and “b” have been quantified (normalized to 18 s RNA levels).
(D) Detection of chimeric miR-451-PKA-R transcripts with ribonuclease protection assays (RPAs) with RNA samples from uninfected whole blood (WB) and purified parasites from HbAA and HbSS erythrocytes. RNA was hybridized to a PKA-R (PKAR) or miR-451-PKAR (451-PKAR) probe, digested with RNaseA/T1, and run on gel. Additionally, one RNA sample was prehybridized to miR-451 DNA probe, digested with RNaseH, subsequently hybridized with a 451-PKA-R probe, and digested with RNaseA/T1 (lane 4). As a control, undigested miR-451-PKA-R probe was included (NR). A detailed schematic of this strategy is shown in Figure S3.
(E) Detection of chimeric miR-451-PEAMT with RPA as in (D).
See also Figure S3 and Tables S2 and S3.
Cell Host & Microbe  , DOI: ( /j.chom )
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5. Figure 4 Cellular Levels of miR-451 Fusion Transcripts
(A) Level of miR-451-PKA-R transcripts in parasites grown in HbAA, HbAS, and HbSS cells transfected with the indicated miRNA or antisense 2′O-me miRNA. Level is expressed as the percentage degradation of PKA-R transcripts after hybridization to a miR-451-PKA-R junction probe followed by RNaseH digestion. The remaining PKA-R transcript levels was determined by real-time PCR and normalized against an RNaseH digestion with a hypothetical miR-181-PKA-R probe (n = 4; mean ± SEM).
(B) Levels of miR-451 modified PKA-R and PEAMT as determined by qPCR. Treatments were identical to (A) (n = 7; mean ± SEM).
(C) miR-451-PKA-R levels within HbAS erythrocytes as determined in (B). n = 6; (mean ± SEM).
(D) Levels of unmodified transcripts (Plasmepsin IV, Falcipain 3, and Rhop2) and miR-451-modified transcripts (PKA-R, and PEAMT) captured by unmodified miR-451, desthiobiotin (Db) miR-181, and Db-miR-451 (normalized versus 18S rRNA, n = 5; mean ± SEM).
(E) Levels of PKA-R enriched by desthiobiotin pull-downs from parasites grown in erythrocytes transfected with miR-451 (Mock), Db-miR-181 (miR-181), or Db-miR-451 (miR-451) in the presence or absence of excess Db-miR-451 (+DbmiR-451 samples) added to the parasite lysate prior to biotin pull-down (normalized versus 18S rRNA, n = 4; mean ± SEM).
(F–H) Levels of miR-451 modified PKA-R (F) and PEAMT (G), as well as parasitemia (H) after erythrocyte transfection with unmodified miR-451 and miR-181, or with 5′ or 3′ Biotin (Bi)- and Phosphate (P)-labeled miR-451 (n = 4; mean ± SEM).
See also Figure S4.
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6. Figure 5
miR-451 Modified Transcripts Are Uncapped and Exhibit Decreased Ribosomal Loading
(A) RPA for detection of miR-451-PKA-R. RPA of terminator exonuclease untreated and treated RNA from parasites grown in HbAA and uninfected whole blood (WB), with a directly synthesized 27 nt RNA probe.
(B) qPCR of terminator exonuclease-treated RNA in both HbAA and HbSS derived parasites. Data is presented in a ratio of undigested/digested, and normalized against β-actin (n = 3; mean ± SEM).
(C) Polysome profile of synchronized parasites at 32 hr after infection.
(D) 18S and 28S P. falciparum rRNA distribution in the gradient fractions. The relative migration of the small and large ribosomal subunits and the 80S monosome are indicated by the ∗, †, and ‡ symbols, respectively.
(E) Normalized levels of total and miR-451-modified PKA-R across the HbAA polysome gradient (n = 4; mean ± SEM).
(F) Relative expression of miR-451-PKA-R across polysome gradients of HbAA and HbSS cells transfected with or without miR-451 (HbAA) or antisense 2′OMe-miR-451 (HbSS).
(G and H) Levels of total PKA-R (G) and Plasmepsin IV (H) across polysome gradients of HbAA and HbSS cells transfected with or without miR-451 (HbAA) or antisense 2′OMe-miR-451 (HbSS) (n = 3 except HbSS-anti miR-451, where n = 1; mean ± SEM).
(I and J) Levels of PKA-R (I) and Plasmepsin IV (J) in fractions 1–17 versus fractions 18+ for HbAA and HbSS cells under the indicated treatments (mean ± SEM).
See also Figure S5.
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7. Figure 6
High miR-451 Levels Result in Decreased PKA-R Protein Levels and Increased Number of Sexual Stage Parasites
(A) PKA-R protein levels in parasites grown in HbAA, HbAS, and HbSS cells that were control treated or transfected with miR-451 or 2′OMe-miR-451. Normalized ratio of PKA-R to Plasmepsin IV (PLAS IV) is also indicated.
(B) Normalized percentage (against mock-transfected) of sexual stage parasites grown in the indicated erythrocyte types, shown at 6 days after transfection, as determined by Giemsa staining (n = 3; mean ± SEM).
See also Figure S6.
Cell Host & Microbe  , DOI: ( /j.chom )
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